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Helper factors derived from autologous mixed lymphocyte cultures
CELLULAR
IMMUNOLOGY
Helper
Factors
DAVID
50, 305-313
Derived
(1980)
from Autologous Cultures
T. Y. Yu, NICHOLAS CHIORAZZI, The Rockqfeller
University,
Rrl'eired
Mov
Mixed
Lymphocyte
AND HENRY G. KUNKEL
New
York
1002i
10. 1979
When human peripheral blood T lymphocytes were cultured with irradiated autologous trinitrophenyl modified autologous cells, cytotoxic cells were generated against the latter, provided that an additional stimulatory signal was also introduced into the cultures. Experiments carried out in this paper showed that this stimulus could be provided by the autologous mixed lymphocyte reaction derived by adding to the cultures autologous non-T mononuclear cells or autologous lymphoblastoid cell line cells. Cell-free supematants obtained from such autologous mixed lymphocyte cultures were also effective. In addition, autologous mixed lymphocyte culture supernatants induced the generation of antibody producing cells from monocyte-depleted peripheral blood mononuclear cells. However, in contrast to the cytotoxic response, this effector function could be induced to a significant degree in the absence of antigen. Quantitation of the percentage of plasma cells formed/cultured was the best indicator of this polyclonal activation. These experiments indicate that helper factors are produced in autologous mixed lymphocyte cultures which can provide a signal to induce the development of cytotoxic and antibody-producing cells.
INTRODUCTION When human lymphocytes from one subject are cultured with those from another, a proliferative reaction occurs. This mixed lymphocyte reaction is accepted as an in vitro model for the in vivo rejection of surgically transplanted tissues (I). An analogous reaction occurs when peripheral blood T lymphocytes are cultured with autologous non-T mononuclear cells or lymphoblastoid cell line cells (2-4). As in the former, this autologous mixed lymphocyte reaction is relatively specific. T cells which have been “primed” by culturing with stimulatory cc!:. beyond the peak of their proliferative reaction respond best in a secondary culture to challenges with stimulatory cells from the original subjects (5, 6). The physiological significance of this reaction is much fess clear. In a previous study (7), it was reported that in an autologous mixed lymphocyte reaction specific cytotoxic cells analogous to those found in an allogeneic mixed lymphocyte reaction were not observed. However, the autologous reaction was found to facilitate the production of cytotoxic cells to heated allogeneic stimulatory cells that did not by themselves stimulate such cells. In this paper, we extend this observation to the examination of the cytotoxic lymphocytes formed against trinitrobenzene sulfonic acid (TNP)‘-modified autologous lymphocytes. Special ’ Abbreviations pokeweed mitogen: fetal calf sera; “‘Cr, CTL, cytotoxic T
used: TNP, trinitrobenzene sulfonic acid: SRBC, sheep red blood cells: PWM PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; FCS, radioactive sodium chromate: PFC, plaque-forming cells: HTdr, tritiated thymidine; lymphocytes. 305 0008-8749/80/040305-09$02.00/O
Copyright 0 1980by Academic Press, Inc. All rights of reproduction in any form reserved.
306
YU,CHIORAZZI,ANDKUNKEL
emphasis was placed in this study on the generation of helper factors in the autologous mixed lymphocyte cultures for the production of cytotoxic cells and antibody-producing cells. MATERIALS
AND METHODS
Isolation of lymphocytes. Mononuclear cells (PBMC) were isolated from samples of peripheral venous blood of normal subjects by Ficoll-Hypaque gradient. To isolate T lymphocytes from the PBMC, monocytes were first removed by incubating the preparations with carbonyl iron particles followed by application of a horse-shoe magnet. The monocyte-free preparations were then rosetted with neuraminidase-treated sheep red blood cells (SRBC). After 60 min incubation at 4”C, the rosettes were gently resuspended and centrifuged on Ficoll-Hypaque gradient. The pellet and interface cells consisted of about 95 and 5%, respectively, of SRBC rosette-forming cells and were designated as T- and B-cell preparations. The SRBC were lysed by exposure to buffered ammonium chloride solution. The cell preparations were washed twice with phosphate-buffered saline (PBS) prior to culture. TNP modification. When they were intended to be used as stimulatory cells, the PBMC were washed three times with PBS and resuspended in 0.5 ml of PBS. To this was added 1.0 ml of 10 mM TNP (Nutritional Biochemical Co., Cleveland, Ohio) dissolved in PBS with pH adjusted to 7.4. After 10 min of incubation at 37”C, they were washed twice with 2% human AB sera in PBS and exposed to 2000 R of irradiation. When used as target cells, the PBMC were washed three times with PBS and the cell pellet resuspended directly in 1.Oml of 10 mM TNP. After 10 min incubation at 37°C they were washed twice with RPMI- 10% fetal calf sera (FCS) prior to use. Lymphoblastoid cell lines. Lymphoblastoid cell lines were established from Bcell preparations of normal individuals by S. M. Fu and J. N. Hurley using methods described previously (8). They were cultured in RPMI-10% FCS and fed with media twice a week. Before using them as helper cells, they were first washed with PBS and incubated with 100 pg of mitomycin in 1.0 ml of Hank’s balanced salt solution for 30 min at 37°C. Lymphocyte cultures. Six-day lymphocyte cultures were carried out in roundbottom, multiwell microculture plates (Linbro 76-013-05) in 37°C 5% CO, incubator using as media RPM1 1640 (Microbiological Associates, Walkersville, Md.) supplemented with 0.2 mM glutamine, 100 units/ml penicillin, 100 pg/ml streptomycin, and 10% AB sera. Into each well was aliquoted 2 x 1Ojof each type of peripheral blood cells and 0.2 x IO5lymphoblastoid cell line cells with or without 0.1 ml of culture supernatants. The final volume was adjusted to 0.2 ml. Cells to serve as target cells were cultured separately in plastic flasks (Falcon 3013) at l2 x lo6 cells per milliliter of media. Cytotoxicity assay. Lymphoblastoid cell line cells or cultured PBMC served as target cells. For each experiment, 1-3 x lo6 of them were labeled in 0.3 ml of RPMI-50% FCS at 37°C for 3 hr using 300 PCi of sodium chromate (51Cr, 200-500 Ci/g, New England Nuclear, Boston, Mass.). After washing three times with PBS and, if necessary, modified with TNP, the PBMC and lymphoblastoid cell line cells were resuspended at 5 and 2 x IO4per milliliter, respectively, in RPMI-20% FCS.
HUMAN
Responders
Stimulators
H Tdr (cpm)
AUTOLOGOUS
307
FACTOR “/. 51 Cr Released
T31tt -10 TNn
A
Ax
3,188
A
CX
39,420
TN;-,
I--/-===
h-,
A A
A
TNP-Ax
TNP-A,
+ Cx
1,279
35,047
TNP-A C
b
TN;-A
tl
,
FIG. 1. Effect of “third party” cells on the generation of cytotoxic cells. A = autologous C = “third party” cells; TNP-A = TNP-modified autologous cells; x = irradiated.
cells;
Effector cells were harvested from the microculture plates by Pasteur pipets and adjusted to the required concentrations with RPMI-20% FCS. One-tenth milliliter of each of effector cells and labeled target cells were pipetted into each well of V-shaped-bottom, microculture plates (Cooke LabProduct, Alexandria, Va.). An effector to target cell ratio of 20: 1 was used throughout this study. The plates were centrifuged at 600 rpm for 5 min, incubated at 37°C for 5 hr and centrifuged again at 1400 rpm for 5 min. One-tenth milliliter of supernatant was transferred from each well into a plastic tube (Falcon 2052). The amount of radioactivity in this sample was assessedas counts per min (cpm) by a gamma counter. Test samples were also carried out in which instead of effector cells 0.1 ml of media or a 0.5% NP-40 in PBS was added to each well of labeled cells. The amount of radioactivity in these samples were designated as spontaneous and total release, respectively. In all esperiments, the spontaneous release were less than 25% of the total release. Assays were done in triplicate and results expressed as percentage 51Crrelease by the following formula: cpm released by effector cells - spontaneous release x 100. Total release - spontaneous release Tritiated thymidine incorporation assay. Lymphocyte cultures were carried out as described above. On the 5th day of the culture 2 &i of tritiated thymidine (HTdr, 1.9 CilmM, SchwarziMann, Orangeburg, N.Y.) were added to each well. The cultures were continued for another 24 hr and the cells were harvested onto glass fiber filters by an automatic cell harvester. Each filter was put into a vial containing 3.5 ml of Aquasol- scintillation fluid (New England Nuclear, Boston, Mass.). The amount of radioactivity incorporated was assessed as cpm by a scintillation spectrometer. Assays were done in triplicate. The results were expressed as averages and standard errors of means. The standard errors of means of the HTdr incorporation and cytotoxicity assays were less than 20 and 5% of the means, respectively. These methods of lymphocyte isolation and culture, cytotoxicity, and HTdr incorporation assays were modified from those reported previously (7).
308
YU, CHIORAZZI, Responders
Stimulators
T
T
T
FIG. 2. Effect of cytotoxic cells.
6,
TNP-A,
B,+
TNP-A,
HTdr (cpm)
30,562
5,153
24,491
AND KUNKEL
Target Cells
7. 51 Cr Released
A TNP-A
A TNP-A
A TNP-A
of autologous non-T mononuclear
P
cell preparations
I
on the generation
Generation of culture supernatanrs. Cutlures were carried out in macroculture plates (Linbro FB-16-24 TC) with 2 x lo6 each of responder and stimulatory cells in 2 ml of RPMI-10% AB sera per well. The stimulatory cells in autologous and allogeneic mixed lymphocyte cultures were autologous B preparations and allogeneic PBMC, respectively. The responder cells in both cases were T-cell preparations. After 48 hr of culture in a 5% CO, 37°C incubator, the supernatants were collected, centrifuged at 600 g for 10 min, and passed through 0.45pm millipore filters. They were stored at -20°C. Supernatants from cultures in which only T-cell preparations were present served as controls. Absorption of supernatants with antibodies. Rabbit antibodies to human Ia and immunoglobulins were insolubilized by attachment to protein A-Sepharose 4B beads (10). These were stored at 4°C. They were washed five times with PBS prior to use. Six-tenth milliliters of each was mixed with 1.Oml of culture supernatant and incubated at 4°C for 45 min. The supernatants were passed through 0.45pm millipore filters prior to use. Generation of plaque-forming cells (PFC). Monocyte-free PBMC were cultured in RPMI-5% AB sera with or without the addition of pokeweed mitogen (PWM l/100 final dilution), SRBC, or supernatants from lymphocyte cultures. After 5 days, the number of PFC in each culture was assayed by a modification of the Jerne-Nordin technique. Details of these procedures have been published (9, 10). Stalistics. The results in each group of several experiments were computed into averages + standard errors of means and were compared with those of another group by the Student’s t test. RESULTS Effecf of “Third Party” Cells PBMC which had been cultured for 6 days with autologous TNP-modified PBMC demonstrated negligible cytotoxic activity against TNP-modified target cells. However, significant increase in such activity was observed when such cultures were carried out with the addition of irradiated PBMC from another subject (“third party” cells). In a total of 15experiments, the percentage of 51Crreleased increased
HUMAN
AUTOLOGOUS
309
FACTOR
from 1.8 k 1.3 to 18.6 2 1.6% (P < 0.0005). As expected, such cells also had considerable cytotoxic activity on the “third party” cells. Data in Fig. 1 were representative of these experiments. effect of Autologous
Non-T Cell Preparations
or Lymphohlastoid
Cell Line Cells
When T-cell preparations were cultured with autologous TNP-modified PBMC, no significant generation of cytotoxic cells was observed. Cytotoxic cells with significant activity developed when the cultures were carried out with the addition of irradiated autologous non-T cell preparations. The average percentage of “Cr released from TNP-modified target cells in eight experiments increased from 3.0 k 1.4 to 17.6 k 1.9% (P < 0.0005). In five other experiments, mitomycintreated autologous lymphoblastoid cell line cells were used instead of the non-T cell preparations. The average percentage of “0 released increased from 0. I + 1.0% to 19.4 2 4.1% (P < 0.0025). Figures 2 and 3 were representative of experiments of each type. The addition of these autologous cells led to a mixed lymphocyte reaction in the responder cells and hence increases in the amount of HTdr incorporated. “Cold inhibition” experiments were carried out to examine the specificity of these cytotoxic cells. Unlabeled, TNP-modified PBMC were added as “cold target” cells to the cytotoxicity assays. This led to a significant decrease in the percentage of “‘Cr released. The inhibition could not be achieved by adding unmodified PBMC (Table 1). This confirmed that the cytotoxic cells were directed against TNP modified but not unmodified target cells. &ffect of Cuiturr
Suprmattints
Supernatants from allogeneic mixed lymphocyte cultures had the same effect as “third party” cells (Fig. 4). Seven experiments were also carried out using supernatants from autologous, mixed lymphocyte cultures plus TNP-modified stimulatory cells to generate cytotoxic cells (Fig. 5). The percentage ;“Cr released increased from 1.6 k 1.3 to 19.4 k 1.6% with the addition of the supernatants in the Responders
Stimulators
H Tdr (cpm)
Target Cells
Ax
687
h TNP-A
A
% 51Cr
Released
LB
A
LBm
11,273
A TNP-A LB
A
TNP-A,
4,148
A TNP-A LB
A
LB,,,+
TNP-Ax
14,789
A TNP-A LB
FIG. 3. Effect of autologous lymphoblastoid cell line cells on the generation LB = lymphoblastoid cell line cells; m = mitomycin treated.
of cytotoxic
cells.
310
YU, CHLORAZZT,
AND KUNKEL
TABLE
1
Effect of Cold Target Cells on the Cytotoxicity Number of cold target cells per well
Cold target cells
20 x 2 x 20 x 2 x
None TNP-A” TNP-A A’ A
% “‘Cr released
104 104 104 104
’ Each well also contained 1 x IO4 “‘Cr-labeled b TNP-modified autologous cells. ’ Unmodified autologous cells.
Assay”
Expt 1
Expt 2
18.4 -0.4
17.1 - 1.7
0.2 17.4 14.0
-0.4 17.2 16.8
target cells and 20 x lo4 unlabeled effector
cells.
cultures (P < 0.0005). Addition of the supernatants alone without the stimulatory cells did not lead to appearance of cytotoxic cells. The supernatants also induced a slight but inconsistent proliferative reaction in the responder cells even in the absence of stimulatory cells. Supernatants from cultures containing T-cell preparations were inactive. Preabsorption of the supernatants with insolubilized anti-Ia or anti-Ig antibodies did not abolish their activity. Effect of Supernatants on the Generation of Antibody Responses As reported previously (lo), PBMC cultured with SRBC alone failed to generate PFC after 5 days of culture. However, with the addition of supernatants from autologous mixed lymphocytes cultures, significant numbers of specific PFC were produced (Table 2). This activation to antibody production was confirmed by enumerating the number of plasma cells produced. In both experiments presented, autologous factors induced a greater than 40-fold increase in antibody synthesizing cells either in the presence or absence of SRBC. Thus the helper factors resulted in the differentiation of a significant number of peripheral blood B cells without the requirement for antigen. This polyclonal response was reflected in the specific PFC response in about 1%of the individuals studied. Responders
Stimulators
Factor
HTdr (cpm)
A
AX
-
1,816
A
AX
+
5,561
Target Cd I *
“/ 51 Cr Released
A
A
TNP-Ax
-
A
TNP-Ax
+
FIG. 4. Effect of allogeneic
7,790
TNP-A
A TNP-A
factor on the generation
of cytotoxic
I
cells. + = present: - = absent.
HUMAN
Respor~ders
Stimulators
A
Ax
A
fi. x
AUTOLOGOUS HTdr (cpm)
Factor
Control Factor
-
-
1,251
+
-
5,274
311
FACTOR Target Cells
% 51Cr
Released
A TNP-A
A
TNP-A,
-
-
A
1,071
TNP-A
A
TNP-A,
-
+
0
A
1,392
TNP-A
A
TNP-A,
+
-
6,971 TNP-A A
FIG. 5. Effect of autologous
factor on the generation
+
of cytotoxic
cells.
DISCUSSION The activities of antigen-specific cytotoxic T lymphocytes (CTL) appear to constitute a major immune defence against viral infections (11) and are perhaps partly responsible for the rejection of certain tumors (12) and development of some experimental autoimmune conditions (13). A frequently used in virvo model to simulate such reactions is the generation of CTL against TNP-modified lymphocytes. This occurs readily when murine splenic lymphocytes are cultured with syngeneic TNP-modified splenic cells (14, 15). In contrast, human PBMC demonstrate a feeble reaction in the primary cultures. However, substantial activity is observed if the cells derived from such primary cultures are rechallenged in a secondary culture with the stimulatory cells (16, 17). An alternate method is to add to the primary cultures irradiated lymphocytes from another subject (“third party” cells) or soluble antigens to which the responder cells are sensitive (18, 19). TABLE Effect of Autologous
2
Factor on Antibody
Production”
Exp. 1
Samples
Factor
Direct anti-SRBC PFCiculture
Exp. 2 9%plasma cells/ culture
Direct anti-SRBC PFCiculture
% plasma cells/ culture
Control Control
+
(5 IO
co.1 7.2
<5 76
co.1 4.3
SRBC SRBC
+
<5 85
co.1 6.8
<5 100
0.2 4.5
PWM PWM
+
15 25
7.4 8.1
115 335
4.6 5.1
a Control samples were those in which monocyte-free PBMC were cultured alone. SRBC samples were those in which 1 x 10fi SRBC were added to each culture. PWM samples were those in which PWM was added at a final dilution of 1:lOO. Each culture contained 3.5 X lo6 cells. PBMC had been depleted of monocytes by the carbonyl iron ingestion technique prior to culture.
312
YU, CHIORAZZI,
AND KUNKEL
In the present study, we confirmed the helper effect of adding “third party” cells and demonstrated that the “third party” cells could be substituted by autologous non-T cell preparations as well as autologous lymphoblastoid cell line cells. Apparently, the autologous mixed lymphocyte reaction which occurred in these cultures was able to promote the generation of antigen-specific cytotoxic cells. The helper effect was also observed using supernatants from the autologous mixed lymphocyte cultures. The extent of the help appeared to approach that obtained from allogeneic cultures. A large variety of factors have been described from allogeneic supernatants. These include the so-called “cell-free media of mixed lymphocyte cultures” (CFM) which induces human lymphocytes to generate cytotoxic T cells directed against phytohemagglutinin-stimulated lymphocytes (20); the killer-assisting factor (KAF) which promotes the generation of murine cytotoxic lymphocytes against heated allogeneic stimulatory cells or sygeneic tumors (21); the lymphocyte mitogenic factor (LMF) which leads to the proliferation of lymphocytes (22); and the allogeneic effect factor (AEF) which in the murine species replaces the requirement for helper T cells in in vitro antibody responses (23, 24). We have recently described a strong AEF from human allogeneic mixed lymphocyte cultures (10,25). The relationship of such factors to one another is not clear. Moreover, since under natural circumstances allogeneic cells are seldom introduced in viva to induce the equivalence of the allogeneic mixed lymphocyte cultures, the physiological significance of these factors have been questioned. In this paper, we demonstrated that factors with some of these properties could be generated in the autologous mixed lymphocyte cultures. The introduction of foreign cells, soluble antigens, or mitogens is not necessary. Although TNP-modified autologous cells are used as a model in this study, the requirement for helper effect to develop CTL is not unique to them. Lymphocytes from patients who are in remission from acute myelogenous leukemia have been induced to develop CTL against autologous cryo-preserved leukemic myeloblasts with the help of “third party” cells (26, 27). The autologous mixed lymphocyte reaction may provide a more physiological way to achieve similar purposes. It may even constitute a natural defense. The fact that the reaction also enhances antibody production strengthens the concept that the reaction may play a modulatory role in immune responses. ACKNOWLEDGMENTS This project was supported by U.S. Public Service Grant AI-1081 1. N. Chiorazzi is a Research Fellow of the Arthritis Foundation.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Bach, F. H., Bach, M. L., and Sondel, P. M., Nature (London) 259, 273, Opelz, G., Kiuchi, M., Takasugi, M., and Terasaki, P., J. Exp. Med. 143, Kuntz, M. M., Innes, .I. B., and Weksler, M. E., J. Exp. Med. 143, 1054, Weksler, M. E., J. C/in. Invest. 51, 3124, 1972. Sheey, M. J., Sondel, P. M., Bach, M. L., and Bach, F. H., Science 188, Weksler, M. E., and Kozak, R., J. Exp. Med. 146, 1833, 1977. VandeStouwe, R. A., Kunkel, H. G., Halper, J. P., and Weksler, M. E., J. 1977. 8. Hurley, J. N., Fu, S. M., Kunkel, H. G., and Mckenna, G., and Scharff, M. Sci. USA 75, 5706, 1978.
1976. 1327, 1976. 1976. 1308, 1975. Exp. Med. 146, 1809, D., Proc. Nut. Acad.
HUMAN
AUTOLOGOUS
FACTOR
313
9. Fauci. A. S.. and Pratt, K. R., J. Eup. !vrd. 144, 674, 1976. 10. Chiorarzi. N., Fu, S. M., and Kunkel, H. G.. f/nmlrnol. Ret,. 45, 1, 1979. 11. Zinkernagel. R. M., and Doherty, P. C., In “Contemporary Topics in Immunobiology” (0. Stutman, Ed.), Vol. 7. Plenum Press, New York, 1977. 12. Allison, A. C.. Cancer Imm~tnol. Immrrnother. 2, 151. 1977. 13. Werke, H., and Begeman, B., Errr. J. Immtrrrol. 8, 294, 1978. 14. Shearer. G. M.. Eltr. J. Immunol. 4, 527. 1974. 15. Shearer, G. M.. and Schmitt-Verhulst, A. M.. Ad~tr~. fmmunol. 25, 55, 1977. 16. Shaw, S., Nelson, D. L., and Shearer. G. M.. .I. Ifr~fnrrnol. 121, 281, 1978. 17. Newman, W., Stoner, C. L., and Bloom, B. R., ,Yrrttrre (London) 269, 151, 1977. 18. Friedman, S. M.. Neyhard, N.. and Chess. L.. J. Immrrnol. 120, 630, 1978. 19. Friedman, S. M., Kuhns. J. Irigoyen, 0.. and Chess, L.. J. Immune/. 122, 1302, 1979. 20. Kasakura, S.. .I. lmmunol. 118, 43, 1977. 21. Fyfe. D. A., and Finke, J. H., .I. Immrrnol. 122, 1156, 1979. 22. Wolstencroft, R. A., Maini, R. N., and Dumonde. D. C., 111“Manual Of Clinical Immunology” (N. R. Rose and H. Friedman. Eds.). American Society for Microbiology, Washington, D. C.. 1976. 23. Armerding, D.. and Katz. D. H., J. E.rp. .Med. 140, 19, 1974. 24. Armerding. D.. and Katz, D. H., J. E.rp. ,Mrd. 140, 1717, 1974. 25. Chiorazzi. N., Fu, S. M., and Kunkel, H. G.. J. E.rp. ,vrd. 149, 1543, 1979. 26. Lee, S. K., and Oliver, R. T. D., .I. I%F. .Mrd. 147, 912, 1978. 27. Zarling, J. M.. Raich, P. C., McKeough. M., and Bach, F. H., Nrrtrrre (Londonj 262, 691, 1976.
IMMUNOLOGY
Helper
Factors
DAVID
50, 305-313
Derived
(1980)
from Autologous Cultures
T. Y. Yu, NICHOLAS CHIORAZZI, The Rockqfeller
University,
Rrl'eired
Mov
Mixed
Lymphocyte
AND HENRY G. KUNKEL
New
York
1002i
10. 1979
When human peripheral blood T lymphocytes were cultured with irradiated autologous trinitrophenyl modified autologous cells, cytotoxic cells were generated against the latter, provided that an additional stimulatory signal was also introduced into the cultures. Experiments carried out in this paper showed that this stimulus could be provided by the autologous mixed lymphocyte reaction derived by adding to the cultures autologous non-T mononuclear cells or autologous lymphoblastoid cell line cells. Cell-free supematants obtained from such autologous mixed lymphocyte cultures were also effective. In addition, autologous mixed lymphocyte culture supernatants induced the generation of antibody producing cells from monocyte-depleted peripheral blood mononuclear cells. However, in contrast to the cytotoxic response, this effector function could be induced to a significant degree in the absence of antigen. Quantitation of the percentage of plasma cells formed/cultured was the best indicator of this polyclonal activation. These experiments indicate that helper factors are produced in autologous mixed lymphocyte cultures which can provide a signal to induce the development of cytotoxic and antibody-producing cells.
INTRODUCTION When human lymphocytes from one subject are cultured with those from another, a proliferative reaction occurs. This mixed lymphocyte reaction is accepted as an in vitro model for the in vivo rejection of surgically transplanted tissues (I). An analogous reaction occurs when peripheral blood T lymphocytes are cultured with autologous non-T mononuclear cells or lymphoblastoid cell line cells (2-4). As in the former, this autologous mixed lymphocyte reaction is relatively specific. T cells which have been “primed” by culturing with stimulatory cc!:. beyond the peak of their proliferative reaction respond best in a secondary culture to challenges with stimulatory cells from the original subjects (5, 6). The physiological significance of this reaction is much fess clear. In a previous study (7), it was reported that in an autologous mixed lymphocyte reaction specific cytotoxic cells analogous to those found in an allogeneic mixed lymphocyte reaction were not observed. However, the autologous reaction was found to facilitate the production of cytotoxic cells to heated allogeneic stimulatory cells that did not by themselves stimulate such cells. In this paper, we extend this observation to the examination of the cytotoxic lymphocytes formed against trinitrobenzene sulfonic acid (TNP)‘-modified autologous lymphocytes. Special ’ Abbreviations pokeweed mitogen: fetal calf sera; “‘Cr, CTL, cytotoxic T
used: TNP, trinitrobenzene sulfonic acid: SRBC, sheep red blood cells: PWM PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; FCS, radioactive sodium chromate: PFC, plaque-forming cells: HTdr, tritiated thymidine; lymphocytes. 305 0008-8749/80/040305-09$02.00/O
Copyright 0 1980by Academic Press, Inc. All rights of reproduction in any form reserved.
306
YU,CHIORAZZI,ANDKUNKEL
emphasis was placed in this study on the generation of helper factors in the autologous mixed lymphocyte cultures for the production of cytotoxic cells and antibody-producing cells. MATERIALS
AND METHODS
Isolation of lymphocytes. Mononuclear cells (PBMC) were isolated from samples of peripheral venous blood of normal subjects by Ficoll-Hypaque gradient. To isolate T lymphocytes from the PBMC, monocytes were first removed by incubating the preparations with carbonyl iron particles followed by application of a horse-shoe magnet. The monocyte-free preparations were then rosetted with neuraminidase-treated sheep red blood cells (SRBC). After 60 min incubation at 4”C, the rosettes were gently resuspended and centrifuged on Ficoll-Hypaque gradient. The pellet and interface cells consisted of about 95 and 5%, respectively, of SRBC rosette-forming cells and were designated as T- and B-cell preparations. The SRBC were lysed by exposure to buffered ammonium chloride solution. The cell preparations were washed twice with phosphate-buffered saline (PBS) prior to culture. TNP modification. When they were intended to be used as stimulatory cells, the PBMC were washed three times with PBS and resuspended in 0.5 ml of PBS. To this was added 1.0 ml of 10 mM TNP (Nutritional Biochemical Co., Cleveland, Ohio) dissolved in PBS with pH adjusted to 7.4. After 10 min of incubation at 37”C, they were washed twice with 2% human AB sera in PBS and exposed to 2000 R of irradiation. When used as target cells, the PBMC were washed three times with PBS and the cell pellet resuspended directly in 1.Oml of 10 mM TNP. After 10 min incubation at 37°C they were washed twice with RPMI- 10% fetal calf sera (FCS) prior to use. Lymphoblastoid cell lines. Lymphoblastoid cell lines were established from Bcell preparations of normal individuals by S. M. Fu and J. N. Hurley using methods described previously (8). They were cultured in RPMI-10% FCS and fed with media twice a week. Before using them as helper cells, they were first washed with PBS and incubated with 100 pg of mitomycin in 1.0 ml of Hank’s balanced salt solution for 30 min at 37°C. Lymphocyte cultures. Six-day lymphocyte cultures were carried out in roundbottom, multiwell microculture plates (Linbro 76-013-05) in 37°C 5% CO, incubator using as media RPM1 1640 (Microbiological Associates, Walkersville, Md.) supplemented with 0.2 mM glutamine, 100 units/ml penicillin, 100 pg/ml streptomycin, and 10% AB sera. Into each well was aliquoted 2 x 1Ojof each type of peripheral blood cells and 0.2 x IO5lymphoblastoid cell line cells with or without 0.1 ml of culture supernatants. The final volume was adjusted to 0.2 ml. Cells to serve as target cells were cultured separately in plastic flasks (Falcon 3013) at l2 x lo6 cells per milliliter of media. Cytotoxicity assay. Lymphoblastoid cell line cells or cultured PBMC served as target cells. For each experiment, 1-3 x lo6 of them were labeled in 0.3 ml of RPMI-50% FCS at 37°C for 3 hr using 300 PCi of sodium chromate (51Cr, 200-500 Ci/g, New England Nuclear, Boston, Mass.). After washing three times with PBS and, if necessary, modified with TNP, the PBMC and lymphoblastoid cell line cells were resuspended at 5 and 2 x IO4per milliliter, respectively, in RPMI-20% FCS.
HUMAN
Responders
Stimulators
H Tdr (cpm)
AUTOLOGOUS
307
FACTOR “/. 51 Cr Released
T31tt -10 TNn
A
Ax
3,188
A
CX
39,420
TN;-,
I--/-===
h-,
A A
A
TNP-Ax
TNP-A,
+ Cx
1,279
35,047
TNP-A C
b
TN;-A
tl
,
FIG. 1. Effect of “third party” cells on the generation of cytotoxic cells. A = autologous C = “third party” cells; TNP-A = TNP-modified autologous cells; x = irradiated.
cells;
Effector cells were harvested from the microculture plates by Pasteur pipets and adjusted to the required concentrations with RPMI-20% FCS. One-tenth milliliter of each of effector cells and labeled target cells were pipetted into each well of V-shaped-bottom, microculture plates (Cooke LabProduct, Alexandria, Va.). An effector to target cell ratio of 20: 1 was used throughout this study. The plates were centrifuged at 600 rpm for 5 min, incubated at 37°C for 5 hr and centrifuged again at 1400 rpm for 5 min. One-tenth milliliter of supernatant was transferred from each well into a plastic tube (Falcon 2052). The amount of radioactivity in this sample was assessedas counts per min (cpm) by a gamma counter. Test samples were also carried out in which instead of effector cells 0.1 ml of media or a 0.5% NP-40 in PBS was added to each well of labeled cells. The amount of radioactivity in these samples were designated as spontaneous and total release, respectively. In all esperiments, the spontaneous release were less than 25% of the total release. Assays were done in triplicate and results expressed as percentage 51Crrelease by the following formula: cpm released by effector cells - spontaneous release x 100. Total release - spontaneous release Tritiated thymidine incorporation assay. Lymphocyte cultures were carried out as described above. On the 5th day of the culture 2 &i of tritiated thymidine (HTdr, 1.9 CilmM, SchwarziMann, Orangeburg, N.Y.) were added to each well. The cultures were continued for another 24 hr and the cells were harvested onto glass fiber filters by an automatic cell harvester. Each filter was put into a vial containing 3.5 ml of Aquasol- scintillation fluid (New England Nuclear, Boston, Mass.). The amount of radioactivity incorporated was assessed as cpm by a scintillation spectrometer. Assays were done in triplicate. The results were expressed as averages and standard errors of means. The standard errors of means of the HTdr incorporation and cytotoxicity assays were less than 20 and 5% of the means, respectively. These methods of lymphocyte isolation and culture, cytotoxicity, and HTdr incorporation assays were modified from those reported previously (7).
308
YU, CHIORAZZI, Responders
Stimulators
T
T
T
FIG. 2. Effect of cytotoxic cells.
6,
TNP-A,
B,+
TNP-A,
HTdr (cpm)
30,562
5,153
24,491
AND KUNKEL
Target Cells
7. 51 Cr Released
A TNP-A
A TNP-A
A TNP-A
of autologous non-T mononuclear
P
cell preparations
I
on the generation
Generation of culture supernatanrs. Cutlures were carried out in macroculture plates (Linbro FB-16-24 TC) with 2 x lo6 each of responder and stimulatory cells in 2 ml of RPMI-10% AB sera per well. The stimulatory cells in autologous and allogeneic mixed lymphocyte cultures were autologous B preparations and allogeneic PBMC, respectively. The responder cells in both cases were T-cell preparations. After 48 hr of culture in a 5% CO, 37°C incubator, the supernatants were collected, centrifuged at 600 g for 10 min, and passed through 0.45pm millipore filters. They were stored at -20°C. Supernatants from cultures in which only T-cell preparations were present served as controls. Absorption of supernatants with antibodies. Rabbit antibodies to human Ia and immunoglobulins were insolubilized by attachment to protein A-Sepharose 4B beads (10). These were stored at 4°C. They were washed five times with PBS prior to use. Six-tenth milliliters of each was mixed with 1.Oml of culture supernatant and incubated at 4°C for 45 min. The supernatants were passed through 0.45pm millipore filters prior to use. Generation of plaque-forming cells (PFC). Monocyte-free PBMC were cultured in RPMI-5% AB sera with or without the addition of pokeweed mitogen (PWM l/100 final dilution), SRBC, or supernatants from lymphocyte cultures. After 5 days, the number of PFC in each culture was assayed by a modification of the Jerne-Nordin technique. Details of these procedures have been published (9, 10). Stalistics. The results in each group of several experiments were computed into averages + standard errors of means and were compared with those of another group by the Student’s t test. RESULTS Effecf of “Third Party” Cells PBMC which had been cultured for 6 days with autologous TNP-modified PBMC demonstrated negligible cytotoxic activity against TNP-modified target cells. However, significant increase in such activity was observed when such cultures were carried out with the addition of irradiated PBMC from another subject (“third party” cells). In a total of 15experiments, the percentage of 51Crreleased increased
HUMAN
AUTOLOGOUS
309
FACTOR
from 1.8 k 1.3 to 18.6 2 1.6% (P < 0.0005). As expected, such cells also had considerable cytotoxic activity on the “third party” cells. Data in Fig. 1 were representative of these experiments. effect of Autologous
Non-T Cell Preparations
or Lymphohlastoid
Cell Line Cells
When T-cell preparations were cultured with autologous TNP-modified PBMC, no significant generation of cytotoxic cells was observed. Cytotoxic cells with significant activity developed when the cultures were carried out with the addition of irradiated autologous non-T cell preparations. The average percentage of “Cr released from TNP-modified target cells in eight experiments increased from 3.0 k 1.4 to 17.6 k 1.9% (P < 0.0005). In five other experiments, mitomycintreated autologous lymphoblastoid cell line cells were used instead of the non-T cell preparations. The average percentage of “0 released increased from 0. I + 1.0% to 19.4 2 4.1% (P < 0.0025). Figures 2 and 3 were representative of experiments of each type. The addition of these autologous cells led to a mixed lymphocyte reaction in the responder cells and hence increases in the amount of HTdr incorporated. “Cold inhibition” experiments were carried out to examine the specificity of these cytotoxic cells. Unlabeled, TNP-modified PBMC were added as “cold target” cells to the cytotoxicity assays. This led to a significant decrease in the percentage of “‘Cr released. The inhibition could not be achieved by adding unmodified PBMC (Table 1). This confirmed that the cytotoxic cells were directed against TNP modified but not unmodified target cells. &ffect of Cuiturr
Suprmattints
Supernatants from allogeneic mixed lymphocyte cultures had the same effect as “third party” cells (Fig. 4). Seven experiments were also carried out using supernatants from autologous, mixed lymphocyte cultures plus TNP-modified stimulatory cells to generate cytotoxic cells (Fig. 5). The percentage ;“Cr released increased from 1.6 k 1.3 to 19.4 k 1.6% with the addition of the supernatants in the Responders
Stimulators
H Tdr (cpm)
Target Cells
Ax
687
h TNP-A
A
% 51Cr
Released
LB
A
LBm
11,273
A TNP-A LB
A
TNP-A,
4,148
A TNP-A LB
A
LB,,,+
TNP-Ax
14,789
A TNP-A LB
FIG. 3. Effect of autologous lymphoblastoid cell line cells on the generation LB = lymphoblastoid cell line cells; m = mitomycin treated.
of cytotoxic
cells.
310
YU, CHLORAZZT,
AND KUNKEL
TABLE
1
Effect of Cold Target Cells on the Cytotoxicity Number of cold target cells per well
Cold target cells
20 x 2 x 20 x 2 x
None TNP-A” TNP-A A’ A
% “‘Cr released
104 104 104 104
’ Each well also contained 1 x IO4 “‘Cr-labeled b TNP-modified autologous cells. ’ Unmodified autologous cells.
Assay”
Expt 1
Expt 2
18.4 -0.4
17.1 - 1.7
0.2 17.4 14.0
-0.4 17.2 16.8
target cells and 20 x lo4 unlabeled effector
cells.
cultures (P < 0.0005). Addition of the supernatants alone without the stimulatory cells did not lead to appearance of cytotoxic cells. The supernatants also induced a slight but inconsistent proliferative reaction in the responder cells even in the absence of stimulatory cells. Supernatants from cultures containing T-cell preparations were inactive. Preabsorption of the supernatants with insolubilized anti-Ia or anti-Ig antibodies did not abolish their activity. Effect of Supernatants on the Generation of Antibody Responses As reported previously (lo), PBMC cultured with SRBC alone failed to generate PFC after 5 days of culture. However, with the addition of supernatants from autologous mixed lymphocytes cultures, significant numbers of specific PFC were produced (Table 2). This activation to antibody production was confirmed by enumerating the number of plasma cells produced. In both experiments presented, autologous factors induced a greater than 40-fold increase in antibody synthesizing cells either in the presence or absence of SRBC. Thus the helper factors resulted in the differentiation of a significant number of peripheral blood B cells without the requirement for antigen. This polyclonal response was reflected in the specific PFC response in about 1%of the individuals studied. Responders
Stimulators
Factor
HTdr (cpm)
A
AX
-
1,816
A
AX
+
5,561
Target Cd I *
“/ 51 Cr Released
A
A
TNP-Ax
-
A
TNP-Ax
+
FIG. 4. Effect of allogeneic
7,790
TNP-A
A TNP-A
factor on the generation
of cytotoxic
I
cells. + = present: - = absent.
HUMAN
Respor~ders
Stimulators
A
Ax
A
fi. x
AUTOLOGOUS HTdr (cpm)
Factor
Control Factor
-
-
1,251
+
-
5,274
311
FACTOR Target Cells
% 51Cr
Released
A TNP-A
A
TNP-A,
-
-
A
1,071
TNP-A
A
TNP-A,
-
+
0
A
1,392
TNP-A
A
TNP-A,
+
-
6,971 TNP-A A
FIG. 5. Effect of autologous
factor on the generation
+
of cytotoxic
cells.
DISCUSSION The activities of antigen-specific cytotoxic T lymphocytes (CTL) appear to constitute a major immune defence against viral infections (11) and are perhaps partly responsible for the rejection of certain tumors (12) and development of some experimental autoimmune conditions (13). A frequently used in virvo model to simulate such reactions is the generation of CTL against TNP-modified lymphocytes. This occurs readily when murine splenic lymphocytes are cultured with syngeneic TNP-modified splenic cells (14, 15). In contrast, human PBMC demonstrate a feeble reaction in the primary cultures. However, substantial activity is observed if the cells derived from such primary cultures are rechallenged in a secondary culture with the stimulatory cells (16, 17). An alternate method is to add to the primary cultures irradiated lymphocytes from another subject (“third party” cells) or soluble antigens to which the responder cells are sensitive (18, 19). TABLE Effect of Autologous
2
Factor on Antibody
Production”
Exp. 1
Samples
Factor
Direct anti-SRBC PFCiculture
Exp. 2 9%plasma cells/ culture
Direct anti-SRBC PFCiculture
% plasma cells/ culture
Control Control
+
(5 IO
co.1 7.2
<5 76
co.1 4.3
SRBC SRBC
+
<5 85
co.1 6.8
<5 100
0.2 4.5
PWM PWM
+
15 25
7.4 8.1
115 335
4.6 5.1
a Control samples were those in which monocyte-free PBMC were cultured alone. SRBC samples were those in which 1 x 10fi SRBC were added to each culture. PWM samples were those in which PWM was added at a final dilution of 1:lOO. Each culture contained 3.5 X lo6 cells. PBMC had been depleted of monocytes by the carbonyl iron ingestion technique prior to culture.
312
YU, CHIORAZZI,
AND KUNKEL
In the present study, we confirmed the helper effect of adding “third party” cells and demonstrated that the “third party” cells could be substituted by autologous non-T cell preparations as well as autologous lymphoblastoid cell line cells. Apparently, the autologous mixed lymphocyte reaction which occurred in these cultures was able to promote the generation of antigen-specific cytotoxic cells. The helper effect was also observed using supernatants from the autologous mixed lymphocyte cultures. The extent of the help appeared to approach that obtained from allogeneic cultures. A large variety of factors have been described from allogeneic supernatants. These include the so-called “cell-free media of mixed lymphocyte cultures” (CFM) which induces human lymphocytes to generate cytotoxic T cells directed against phytohemagglutinin-stimulated lymphocytes (20); the killer-assisting factor (KAF) which promotes the generation of murine cytotoxic lymphocytes against heated allogeneic stimulatory cells or sygeneic tumors (21); the lymphocyte mitogenic factor (LMF) which leads to the proliferation of lymphocytes (22); and the allogeneic effect factor (AEF) which in the murine species replaces the requirement for helper T cells in in vitro antibody responses (23, 24). We have recently described a strong AEF from human allogeneic mixed lymphocyte cultures (10,25). The relationship of such factors to one another is not clear. Moreover, since under natural circumstances allogeneic cells are seldom introduced in viva to induce the equivalence of the allogeneic mixed lymphocyte cultures, the physiological significance of these factors have been questioned. In this paper, we demonstrated that factors with some of these properties could be generated in the autologous mixed lymphocyte cultures. The introduction of foreign cells, soluble antigens, or mitogens is not necessary. Although TNP-modified autologous cells are used as a model in this study, the requirement for helper effect to develop CTL is not unique to them. Lymphocytes from patients who are in remission from acute myelogenous leukemia have been induced to develop CTL against autologous cryo-preserved leukemic myeloblasts with the help of “third party” cells (26, 27). The autologous mixed lymphocyte reaction may provide a more physiological way to achieve similar purposes. It may even constitute a natural defense. The fact that the reaction also enhances antibody production strengthens the concept that the reaction may play a modulatory role in immune responses. ACKNOWLEDGMENTS This project was supported by U.S. Public Service Grant AI-1081 1. N. Chiorazzi is a Research Fellow of the Arthritis Foundation.
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1976. 1327, 1976. 1976. 1308, 1975. Exp. Med. 146, 1809, D., Proc. Nut. Acad.
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AUTOLOGOUS
FACTOR
313
9. Fauci. A. S.. and Pratt, K. R., J. Eup. !vrd. 144, 674, 1976. 10. Chiorarzi. N., Fu, S. M., and Kunkel, H. G.. f/nmlrnol. Ret,. 45, 1, 1979. 11. Zinkernagel. R. M., and Doherty, P. C., In “Contemporary Topics in Immunobiology” (0. Stutman, Ed.), Vol. 7. Plenum Press, New York, 1977. 12. Allison, A. C.. Cancer Imm~tnol. Immrrnother. 2, 151. 1977. 13. Werke, H., and Begeman, B., Errr. J. Immtrrrol. 8, 294, 1978. 14. Shearer. G. M.. Eltr. J. Immunol. 4, 527. 1974. 15. Shearer, G. M.. and Schmitt-Verhulst, A. M.. Ad~tr~. fmmunol. 25, 55, 1977. 16. Shaw, S., Nelson, D. L., and Shearer. G. M.. .I. Ifr~fnrrnol. 121, 281, 1978. 17. Newman, W., Stoner, C. L., and Bloom, B. R., ,Yrrttrre (London) 269, 151, 1977. 18. Friedman, S. M.. Neyhard, N.. and Chess. L.. J. Immrrnol. 120, 630, 1978. 19. Friedman, S. M., Kuhns. J. Irigoyen, 0.. and Chess, L.. J. Immune/. 122, 1302, 1979. 20. Kasakura, S.. .I. lmmunol. 118, 43, 1977. 21. Fyfe. D. A., and Finke, J. H., .I. Immrrnol. 122, 1156, 1979. 22. Wolstencroft, R. A., Maini, R. N., and Dumonde. D. C., 111“Manual Of Clinical Immunology” (N. R. Rose and H. Friedman. Eds.). American Society for Microbiology, Washington, D. C.. 1976. 23. Armerding, D.. and Katz. D. H., J. E.rp. .Med. 140, 19, 1974. 24. Armerding. D.. and Katz, D. H., J. E.rp. ,Mrd. 140, 1717, 1974. 25. Chiorazzi. N., Fu, S. M., and Kunkel, H. G.. J. E.rp. ,vrd. 149, 1543, 1979. 26. Lee, S. K., and Oliver, R. T. D., .I. I%F. .Mrd. 147, 912, 1978. 27. Zarling, J. M.. Raich, P. C., McKeough. M., and Bach, F. H., Nrrtrrre (Londonj 262, 691, 1976.